Renal Transplantation
Azim Gangji MD FRCPC FACP
McMaster University
Objectives
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Overview of Immunology
Cross match test
Immunosuppressive Medications
Rejection
– Antibody Mediated Rejection
– Acute Cellular Rejection
• Causes of Allograft Failure
• Surgical Complications
• Longterm Medical Complications
What is MHC and What is its Role
• Human Leukocyte Antigen (HLA) system is
synonymous with the human Major
Histocompatibility Complex (MHC)
• In humans, it’s called HLA due to expression of
gene products on surface of WBC
• These terms describe a group of genes on
chromosome 6 that encode a variety of cell
surface markers, antigen presenting molecules
and other proteins involved in immune function
• Inheritance ~ a haplotype from each parent
What is MHC and What is its Role
• HLA region is subdivided into 3 regions:
– Class I
• encodes “classical antigens” HLA A, B, C
• Expressed on all nucleated cells
• Class I alpha genes are coded by the MHC whereas the B microglobulin
gene is coded on chromosome 15 (non MHC)
– Class II
• Encodes HLA- DR, DQ, DP
• Has both alpha and beta genes both of which are coded by MHC genes
• DR genes all share the same alpha chain but vary in the beta chain whereas
DQ and DP can have polymorphic alpha and beta chains
• In addition, the number of DR genes can vary among individuals. For
example, 2 DR molecules are expressed on a single haplotype: both dimers
use the same alpha chain but one uses a beta chain encoded by DRB1 and
the other uses a beta chain encoded by a second DR locus called DRB3,
DRB4, DRB5, etc. Alleles at this locus are usually expressed at much lower
levels on the cell surface.
– Class III
• Contains genes for complement, TNF, Heat shock protein
MHC Molecules
3D structure of MHC Class II
Molecule
Schematic of MHC
• The structure of class I MHC
and
• class II MHC molecules. The
schematic diagrams and
models of the crystal structures
of class I MHC and class II
MHC molecules
• Both types of MHC molecules
contain peptide-binding clefts
and invariant portions that bind
CD8 (the 3 domain of class
I) or CD4 (the 4 domain of
class II).
Comparison of MHC Class I vs II
Comparison of MHC class
I and class II molecules
Class I MHC
Class II MHC
Structure
MHC encoded chain, beta-2
microglobulin
MHC encoded chain, MHC
encoded beta-chain
Antigen binding groove*
Closed
Open
Peptide length
9 to 10 amino acids
12 to 28 amino acids
Source of antigen
Intracellular
Extracellular
Expression
All nucleated cells
B cells, macrophages, dendritic
cells
Antigen presented to:
CD8+ T cells
CD4+ T cells
Direct and Indirect Allorecognition
The Processing of Antigens and
Presentation to T Cells
T Cell Recognition of Alloantigen:
Indirect Pathways
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Intact donor MHC molecules are
also known to be continuously
shed into the circulation where
they may then be endocytosed by
the recipient's antigen-presenting
cells.
Within the acidic endosomal
compartment, the MHC proteins
are fragmented into their
constituent peptides. They are
then transferred into the
endoplasmic reticulum, loaded into
the antigen binding cleft of MHC
class II of the recipient and finally
expressed on the cell surface of
recipients APCs. This mechanism
has been referred to as the
"indirect allorecognition.“
However, this is of course the
normal route by which T cells
normally encounter antigen ie in
context of self MHC.
T Cell Recognition of Alloantigen:
Direct Pathways
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•
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T cell activation by the "direct"
pathway occurs when T cells
encounter intact donor MHC
on the surface of donor
antigen-presenting cells
(APC). Since MHC molecules
w/o a bound peptide are
unstable & unrecognizable by
T cells, endogenous proteins
bound to the donor MHC may
play a role.
Direct allorecognition of intact
surface MHC molecules has
only been demonstrated in
allogenic transplantation
This pathway is thought to be
of dominant importance during
early acute rejection as
engrafted organs contain a
large number of passenger
leukocytes.
The Processing of Antigens and
Presentation to T Cells
C
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A
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I
C
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A
S
S
II
T Cell Receptor and APC
Interaction: Signal 1
• HLA peptide
presentation by APC
to T Cell
• Triggering of the TCR
by antigen initiates a
signaling cascade
started by the
signaling complex
made up of CD3
APC and T Cell Interaction:
Role of Costimulation: Signal 2
Role of Costimulation: Signal 2
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•
Naïve T cells require both signal 1 and
Memory T cells do not require costimulation through CD28 to become
activated. Costimulation through ICOS is sufficient for memory T cell
activation. c, Absence of B7 or blocking of B7 with CTLA-4 Ig leads to
anergy of the T cell.
Allorecognition and Stimulation of T
Cells and Effect
B, T and NK Cells and Effect:
Rejection
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Fig 2. Classes of lymphocytes. Different classes of
lymphocytes recognize distinct types of antigens
and
differentiate into effector cells whose function is to
eliminate the antigens. B lymphocytes recognize
soluble or cell
surface antigens and differentiate into antibodysecreting cells. Helper T lymphocytes recognize
antigens on the
surfaces of APCs and secrete cytokines, which
simulate different mechanisms of immunity and
inflammation.
Cytolytic T lymphocytes recognize antigens on
infected cells and kill these cells. (Note that
lymphocytes recognize
peptides that are displayed by MHC molecules.)
Natural killer cells recognize changes on the surface
of infected
cells and kill these cells. It should be emphasized
that native T cells (CD4 or CD8) are activated by
professional
APCs. Effector CD8 T cells, not native T cells, can
kill and infected cell expressing the specific peptideclass I
complex.
Immune Activation and Rejection
Stimulus
Activation
Immune System
Encounters the
Transplant
Hyperacute
Rejection
0
0.5
1
Expansion
T Cells
T Lymphocytes Divide
Are Activated
Plasma
And Make
Cells
Cytokines
Make
Antibody
2… 24 hours….
Rejection
Effector
T Cells
Attack the
Transplant
Humoral
Rejection
3-4 days…
Memory
Immune
System
Develops
T and B
cell memory
7 days...
Acute Cellular Rejection
T Cell Mediated Rejection
•
T cell receptor (TCR) of alloreactive
cytotoxic lymphocytes (CTL)
recognize allogeneic major
histocompatibility complex (MHC) on
target cells. CTL mobilize cytotoxic
granules containing perforin and
granzyme B (Gz B) toward the target
cell releasing the cytotoxic molecules
into the intercellular space. Perforin
inserts into the target cell membrane
and Gz B binds to its receptor and
both are internalized to induce
apoptosis. TCR stimulation
increases expression of Fas ligand
(FasL) on the CTL surface and binds
the Fas receptor, triggering the
apoptotic cascade. CTL can produce
the cytotoxic cytokine tumor necrosis
factor α (TNF-α), which binds the
TNF-R on the target cell leading to
apoptosis. CTL can also release
interferon (IFN)-γ, which will activate
the macrophage to release
proinflammatory substances. NO,
nitric oxide; ROS, reactive oxygen
species.
T Cell Mediated Rejection on Renal
Biopsy
• Tubulitis
– Infiltration of tubular
epithelium by T
lymphocytes (arrows)
that have crossed the
basement membrane.
• Endothelialitis
– Invasion of the
endothelium of a large
artery by graftinfiltrating lymphocytes
(arrow).
Antibody and Complement
Mediated Cytotoxicity ie AMR
Antibody Mediated Rejection on
Renal Transplant Biopsy
• Evidence of
peritubular capillary
C4d staining c/w AMR
Complement Dependent
Cytotoxicity
CDC Crossmatch
Recipient’s serum + Donor lymphocytes (with defined HLA) + Complement,
Incubate, add Eosin; remember here lymphocytes are just serving as cells
Complement Dependent
Crossmatch
Pre-Transplant
Therapy
Antibody
Suppression
The Phases of
Immunosuppression
Late Acute Rejection
Early Acute Rejection
Maintenance
Immunosuppression
Acute Immune
Desensitization
Chronic Allograft
Dysfunction
Immune
Accommodation
Induction
Therapy
Acute Post-Transplant
Immunosuppression
Graft Failure
Immunosuppressant Action
Overview
•
Three events in T cell
activation
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Signal 1: Engagement of the
T cell receptor with the
antigen peptide in the context
of self major
histocompatibility complex
(MHC) class II molecule
leads to the activation of the
calcineurin pathway and
results in the induction of
cytokine genes (e.g.,
interleukin [IL]-2).
•
Signal 2: The costimulatory
signal, involves the
engagement of CD28 with
members of the B7 family.
This synergizes with signal 1
to induce cytokine
production.
•
Signal 3: Interaction between
cytokine production and its
corresponding receptor leads
to induction of cell division,
probably through the target of
rapamycin pathway. This
constitutes signal 3.
Categories of Agents
• Induction agents
– Monoclonal or polyclonal antibodies
– Administered intravenously in the perioperative period
– Induce acute, powerful, short-lived immunosuppression
• Desensitization agents
– Pretransplant IVIg can desensitize patients’ immunity
to HLA
– Rituximab is gaining interest as a desensitization agent
• Primary immunosuppressants
– CNIs are the cornerstones of immunosuppressive therapy
• Adjuvant agents
– 1 or more medications prescribed in addition to a CNI
Induction Agents
• Non Depleting Antibodies
– Basiliximab
– Daclizumab
• Depleting Antibodies
– Alemtuzumab
– Muromonab-CD3
– Equine polyclonal IgG antibody
– Rabbit polyclonal IgG antibody
Primary Immunosuppressants
Calcineurin Inhibitors (CNIs)
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Calcineurin inhibition
prevents nuclear
factor (NFAT)
activation.
In the absence of
cyclosporine, calcium
activates calcineurin by
exposing its
phosphatase site,
which, in turn,
activates NFAT.
Cyclosporine forms a
complex with
cyclophilin (CP), which
binds to calcineurin
(CN) and sterically
hinders the
phosphatase site.
Mechanism of Action: Tacrolimus
• Calcium-CalmodulinCalcineurin form a
complex to
dephosphorylate
NFAT
• Tacrolimus binds to
FK binding protein
which binds to
calcineurin and
inhibits this process
Is Tacrolimus Just a Clone of
Cyclosporine?
CNI Side Effects
Event1
Hepatotoxicity
Comments
• Tacrolimus = CsA
Cardiovascular
• Less HTN and Hyperlipidemia with
• Hypertension
Tacrolimus
• Hypercholesterolemia
Post Transplant DM
• Tacrolimus causes more post transplant
Diabetes Mellitus
Neurotoxicity
• Tremor
• Headache
• Insomnia
• Paresthesia
• Seen more often with TAC and
generally improve with dose reduction
1. Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.
2. Johnson C, et al. Transplantation. 2000;69:834-841.
3. Margreiter R. Lancet. 2002;359:741-746.
CNI Side Effects
Event
Comments
Cosmetic
• Gingival hypertrophy
• Hirsutism
• Alopecia
• Use of corticosteroids may exaggerate
development
• Gingival hypertrophy and hirsutism are
associated with CsA
• Alopecia can occur with TAC
Malignancy
• Skin cancers
• Cervical cancer
• Lymphoproliferative
disorders
• Incidence appears to be a function of
overall amount and duration of
immunosuppression rather than any
specific agent
Gaston RS. Am J Kidney Dis. 2001;38(suppl):S25-S35.
Metabolic Interactions That
Increase CNI Levels
• Calcium channel blockers
– Verapamil
– Diltiazem
– Nicardipine
• Antifungal agents
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Ketoconazole
Fluconazole
Itraconazole
Clotrimazole
Metronidazole
• Immunosuppressants
– Sirolimus increases CsA levels
• Antibiotics
– Erythromycin
– Clarithromycin
– Azithromycin
• Protease Inhibitors
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Saquinavir
Indinavir
Nelfinavir
Ritonavir
• Foods
– Grapefruit
– Grapefruit juice
Metabolic Interactions That
Decrease CNI Levels
• Antituberculosis
drugs
– Rifampin
– Rifabutin
– Isoniazid
• Anticonvulsants
– Barbiturates
– Phenytoin
– Carbamazepine
• Herbal preparations
– St John’s wort
Mechanism of action of
mycophenolic acid (MPA)
•
Lymphocytes use the do
novo pathway for generation
of purines (guanine).
•
Mycophenolate mofetil
(MMF) is converted in the
liver by ester hydrolysis to
mycophenolic acid which in
turn non-competitively and
reversibly inhibits IMPDH
activity during DNA synthesis
in the S phase of the cell
cycle.
•
In the salvage pathway,
guanine is converted to GMP
by the enzyme
hypoxanthine-guanine
phosphoribosyltransferase
MPA Adverse Reactions
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Bone marrow suppression
n/v/d
Oral and colonic ulcerations
Colitis
Azathioprine
• Is an antimetabolite that
is a purine analogue that
is incorporated into DNA
and halts synthesis
• Inhibits proliferation of T
and B cells and effect is
mediated by AZA
metabolites, 6-MP, 6-TU,
6-MMP, 6-TGN
Azathioprine Adverse Effects
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Bone marrow suppression
Hepatotoxicity
Alopecia
Drug Interactions:
– Allopurinol
Sirolimus/Everolimus
• Sirolimus binds to FK
binding protein but does
NOT inhibit calcineurin.
• Instead Sirolimus inhibits
mTOR and blocks IL-2
mediated cell proliferation
• mTOR activates protein
that trigger cell cylcle G1
to S progression
Sirolimus Adverse Effects
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Hypercholesterolemia
Hypertriglyceridemia
Edema
Hypertension
Rash
Bone marrow suppression
Interstitial pneumonitis
Delayed wound healing
Mouth ulcers
Myalgia/weakness
Drug fever
Proteinuria
Corticosteroids
• Anti inflammatory and
immunosuppressive
effects
• Suppress production of
numerous cytokines (IL-1,
TNF, IL-2, chemokines,
prostaglandins,
proteases, NFK-B)
• Also affect chemotaxis
(neutrophilia)
Corticosteroids Side Effects
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Acne
Cushingoid facial appearance
Hirsutism
Mood disorders
Hypertension
Glucose intolerance
Cataracts
Osteoporosis
Growth retardation in children
Done by your Local Urologist
I always recommend drinking at least 68 glasses of fluid for the procedure!”
Transplant Surgery
Transplant Surgery
Post Surgical Complications
• Vascular complications
– Bleeding from vessels in the hilum
– Anastamosis hemorrhage (more common with
multiple arteries)
– Renal artery thrombosis (due to hypotension
in the OR, technical complications,
antiphosholipid ab syndrome)
– Renal vein thrombosis (usually 3-9 days post
op and related to technical complications,
antiphosholipid ab syndrome)
Post Surgical Complications
• Vascular complications
– Transplant Renal Artery Stenosis
• Atherosclerosis of recipient vessel
• Clamp injury to donor or recipient vascular
endothelium
• Faulty suture technique (primarily seen with endto-end anastomosis
• Angulation due to diproportionate length between
graft artery and iliac artery
• Kinking of the renal artery
Post Surgical Complications
• Lymphocele
– Collection of lymph caused by leakage from several
lymphatics that overlie the iliac vessels
– Usually present weeks after transplantation
– Can lead to ureteral obstruction, compression of iliac
vein and swelling/DVT of leg, incontinence due to
bladder compression, scrotal masses
– Can be avoided by minimizing dissection of iliac
vessels and ligating lymphatics
– Sirolimus increased the incidence of lymphoceles
from 18% to 38% therefore not used as an induction
agent
Post Surgical Complications
• Urine Leaks
– Occur within the first few days
– Due to technical or as a result of ureteral slough due to
disruption or ureteral blood supply. Blood supply to distal ureter
is the most endangered during harvesting
• Ureteral Obstruction
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Technical
Blood clots
Ureteral slough
Ureteral fibrosis
• Ischemia, rejection or polyoma virus BK
– Extrinsic compression
• Kinking or periureteral fibrosis from lymphoceles or graft rejection
Post Surgical Complications
• Wound
– Infections
– Healing becomes a problem in obese
recipients
• GI Complications
– Bowel obstruction, colonic perforation, gastric
ulcer
Delayed Graft Function
• Poor definition:
– Need for dialysis in the 1st week post transplant
• Etiology
– Immune
• Hyperacute rejection
– Non immune
• ATN, intravascular volume contraction
• Technical: arterial, venous or ureteric occlusion, urine leak
• Recurrent disease (HUS/TTP)
Delayed Graft Function
• Factors promoting ischemic injury in Deceased
donor renal transplantation
– Donor Factors (age, h/o HTN, DM, ARF)
– Donor Management (brain-death stress, ionotropes,
nephrotoxins, cardiac arrest)
– Procurement surgery (Hypotension, traction on renal
vessels, flushing solution)
– Kidney storage (pump vs cold storage, prolonged
time)
– Recipient status (volume status, body habitus, pelvic
atherosclerosis, poor cardiac output, hypotension in
OR, preformed antibodies, prolonged warm ischemia
time)
Renal Transplant Program at
McMaster University
• All forms of transplantation
– Standard Criteria
– Donation after Cardiac Death
– Extended Criteria (donors > 60yo, donors 5059yo with 2 additional RF (h/o HTN, death as
a result of a stroke, elevated terminal serum
Cr > 132umols/L)
– Cross Match Positive Transplants
– ABO incompatible Transplants
Rejection and Treatment
• Acute Cellular Rejection
– Steroids, Thymoglobulin, CNI
• Antibody Mediated Rejection
– PLEX
– IVIG
– Rituximab
– Bortezomib
– Urgent splenectomy
Rejection and Treatment
• And of course prayer!
Surgical
resident ?
Longterm Complications
• Allograft Failure
– On average 50% of DDRT allografts are functioning at
the 10-12 year mark
– On average 50% of living donor allografts are
functioning at the 15-17 year mark
– Etiology of Allograft Failure
• Immune: Rejection, HLA mismatch, prior sensitization history,
medication non compliance
• Non Immune: donor factors (age, ECD, etc), CNI toxicity,
HTN, Hyperlipidemia, Infection (CMV), Proteinuria, renal size
mismatch, recurrent disease, BK Nephropathy
Longterm Complications
Longterm Complications
• Renal Tx patients are at high risk of dying
with a functioning allograft
– 40% from CVD
– 25% infection
– 10% malignancy
– 25% other
Longterm Complications
• CVD Risk Factors
– 20% of renal transplant recipients will develop
impaired fasting glucose post tx and 10% will develop
T2DM
– Prior ESRD pts
– HTN
– Hyperlipidemia
– Cigarette smoking
– Inflammation
– Infections (CMV)
Longterm Complications
• Infections
– CMV
– BK
– Hepatitis C
– Influenza
– Fungal
Infection Timeline Post Transplant
Fishman NEJM 2007
Malignancy
• Skin Cancer: 3.5 fold increase
• Post Transplant Lymphoproliferative
disorder
• GU cancers ~HPV related vulva, vagina,
cervical cancers
• Liver cancer (Hep B, C pts)
• Cumulative incidence of malignant tumors
over 30 years is 33%
Summary
• Overview of Immunology
• Immunosuppressive Medications
– Know pharmacology and adverse effects
• Rejection
– Antibody Mediated Rejection
– Acute Cellular Rejection
• Causes of Allograft Failure: differentiate immune
vs non immune
• Surgical Complications
• Longterm Medical Complications
Thank You For Your Attention
• Causes of Elevated
Resistive Index
• Parenchymal
– Acute Rejection
– Acute Tubular Necrosis
– Pyelonephritis
• Vascular
– Renal Vein Thrombosis
– Hypotension
• Urological
– Ureteral Obstruction
• Technical
– Graft Compression